(1) Field of the Invention
The present invention relates to methods of modulating gene expression in plants and to novel proteins and nucleotide sequences, and in particular, to methods of using such novel proteins and nucleotides for regulating the effects of post transcriptional gene silencing in plants.
(2) Description of the Related Art
With the advent of recombinant DNA technology in the 1970s, the genetic manipulation of plants entered a new age. Genes and traits previously unavailable through traditional breeding became available through DNA recombination, and with greater specificity than ever before. Commercially important genes from sexually incompatible plants, animals, bacteria or insects can now be successfully introduced into plants. Products of modern plant genetic engineering are already on the market, and examples include such transgenic plants as slow-softening tomatoes, cotton and corn plants resistant to herbicides and insects, and soybeans with altered fatty acid profiles. With many more products in the pipeline, the genetic engineering of plants is expected to have a profound impact on the future of agriculture.
Modern plant genetic engineering can involve the transfer of a desired gene into the plant genome and regeneration of a whole plant from the transformed tissue. Unfortunately, many of these efforts have been met with mixed results with regards to predictable and sustainable levels of gene expression.
Many factors affect gene expression in plants. One mechanism, RNA silencing, is an important regulatory mechanism that has only recently been intensively studied. RNA silencing is characterized by reduced levels of the specific mRNA encoded by the silenced gene. Individual cases of silencing include those in which mRNA level is regulated transcriptionally and those in which it is regulated post-transcriptionally.
RNA silencing in transgenic plants was first discovered after genetic transformation of plant tissues with multiple copies of an exogenous transgene that shared some homology with an endogenous gene. Researchers noticed that over time, high levels of exogenous gene expression would unexplainably falter and drop to low levels.
Recently, it has been reported that RNA silencing can be induced by plant virus infections in the absence of any known homology between the viral genome and an endogenous gene. It has been proposed that gene silencing may have evolved as a defense mechanism against viral invasion.
All genomes, including those of plants, animals, and fungi have specific defense mechanisms against infection by foreign bioactive agents. Thus, it has been proposed that RNA silencing is an ancestral mechanism that plant cells use as a defense against invading nucleotide molecules in a sequence specific manner. Therefore, one emerging view is that RNA silencing is part of a sophisticated network of interconnected pathways for cellular defenses (e.g., against viruses and transposons), RNA surveillance, and development, and that it could become a powerful tool to manipulate gene expression.
When viruses or transgenes are introduced into plants, they often trigger a particular host response that is generally referred to as post-transcriptional gene silencing (PTGS) or cosuppression. The mechanism of PTGS is believed to be similar to that of RNA interference (RNAi) in animals. The process appears to be initiated by double-stranded RNA (dsRNA) molecules, which may be generated by replicative intermediates of viral RNAs or by aberrant transgene-coded RNAs (these RNA molecules may become double-stranded when copied and/or amplified by an RNA-dependent RNA polymerase). The dsRNAs are digested into 21-23 nucleotide (nt) small interfering RNAs (siRNA). Evidence indicates that siRNAs are produced when the enzyme Dicer, a member of the RNase III family of dsRNA-specific ribonucleases, digests dsRNA in an ATP-dependent, processive manner. Successive cleavage events degrade the RNA to 19-21 bp duplexes (siRNAs), each with 2-nucleotide 3′ overhangs. The conversion of dsRNA into siRNAs requires additional protein co-factors that may recruit the dsRNA to Dicer or stabilize the siRNA.
These siRNAs direct the degradation of target mRNAs complementary to the siRNA sequence by subsequently binding to a nuclease complex to form what is known as the RNA-induced silencing complex (RISC). Thereafter, the RISC complex binds and destroys the complementary target mRNAs, which in turn, leads to the silenced target RNA phenotype.
As mentioned previously, in both plants and animals, RNA silencing has evolved, at least in part, as an antiviral defense pathway. In response, many viruses have developed genes that encode suppressors of silencing. For example, it has been reported that certain plant viruses encode proteins that can suppress RNA silencing. One suppressor protein in particular, helper component-proteinase (HC-Pro), is produced by a number of plant potyviruses. HC-Pro acts to suppress transgene-induced silencing by interfering with the accumulation of the 21-23 nucleotide siRNAs. It has been found that infection of transgenic plants by viruses expressing HC-Pro can cause wholesale suppression of RNA silencing, resulting in the stabilization of target gene expression. For several reasons, however, it is not desirable to use the viral HC-Pro protein as a vector to suppress silencing. Furthermore, in cases where suppression of silencing is undesirable, actual resistance to HC-Pro suppression is required.
These recent studies on RNA silencing have furthered the understanding of the regulatory mechanisms underlying gene expression. However, In order to better utilize transgenic plants, the mechanisms behind transgene expression and endogenous gene suppression need to be controllable. The ability to quickly and easily create knock out phenotypes using protein components of the RNA silencing pathway would be desirable in terms of plant biotechnology.
From the foregoing, it can be seen that a need exists for improved methods of modulating gene expression in plant cells and plants, and in particular, for methods of regulating—by either reducing or enhancing—the expression of a certain target genes in plants. Such methods are needed to control gene suppression and to obtain acceptable expression levels of genes of interest.